It is proposed to study pig kidney diamine oxidase and bovine aortic lysyl oxidase as part of a long-term aim of comparing the active sites of representative copper-containing amine oxidases. Emphasis will be on clarification of the nature of the active site and of the role played by the copper cofactor in the mechanism of action. The intention is to provide structural bases to help elucidate the detailed mechanisms of action of these enzymes, particularly with respect to substrate specificities and the reoxidation half of the catalytic cycle, and to provide insight into the similarities and differences of the active sites of the different amine oxidases. The work opens a new area of study for lysyl oxidase and extends studies on diamine oxidase, and has significant physiological significance since diamine oxidase regulates neurologically- important amines and lysyl oxidase is required for connective tissue formation and stability. Specific aims are: to characterize the nature of the copper environment by EPR studies in the resting enzymes and in the presence of selected reagents expected to interact with the copper and/or the PQQ; to investigate the hypothesis that the copper is playing an active role in the enzyme mechanisms by removing copper and correlating its reincorporation or replacement by other metals with activity and spectroscopic properties; to probe the polarity and size of the substrate-binding site and to test current models for the active sites of the enzymes, by using spin labels; to measure the distances and magnetic interactions between the copper and the bound substrate using spin- and fluorine-labeled probes and EPR and NMR spectroscopy; to study the pH-site and copper-bound hydroxide act as bases in the mechanism; to determine whether superoxide or hydroxyl radicals are intermediates in the reactions by studying the effect on the activity of small copper complexes known to have superoxide dismutase activity, by spin-trapping techniques to observe radical intermediates, and by rapid- freeze EPR experiments to detect radicals formed during the reaction; and to elucidate the redox behavior of the enzymes using electrochemical techniques.